Impact of bioresorbable versus permanent polymer on longterm vessel wall inflammation and healing: a comparative drug-eluting stent experimental study.

AIMS: Drug-eluting stents (DES) have evolved to using bioresorbable polymers as a method of drug delivery. The impact of bioresorbable polymer on long-term neointimal formation, inflammation, and healing has not been fully characterised. This study aimed to evaluate the biological effect of polymer resorption on vascular healing and inflammation. METHODS AND RESULTS: A comparative DES study was performed in the familial hypercholesterolaemic swine model of coronary stenosis. Permanent polymer DES (zotarolimus-eluting [ZES] or everolimus-eluting [EES]) were compared to bioresorbable polymer everolimus-eluting stents (BP-EES) and BMS. Post implantation in 29 swine, stents were explanted and analysed up to 180 days. Area stenosis was reduced in all DES compared to BMS at 30 days. At 180 days, BP-EES had significantly lower area stenosis than EES or ZES. Severe inflammatory activity persisted in permanent polymer DES at 180 days compared to BP-EES or BMS. Qualitative para-strut inflammation areas (graded as none to severe) were elevated but similar in all groups at 30 days, peaked at 90 days in DES compared to BMS (p<0.05) and, at 180 days, were similar between BMS and BP-EES but were significantly greater in DES. CONCLUSIONS: BP-EES resulted in a lower net long-term reduction in neointimal formation and inflammation compared to permanent polymer DES in an animal model. Further study of the long-term neointima formation deserves study in human clinical trials.

The SYNERGY biodegradable polymer everolimus eluting coronary stent: Porcine vascular compatibility and polymer safety study.

AIMS: SYNERGY is a novel platinum chromium alloy stent that delivers abluminal everolimus from an ultrathin poly-lactide-co-glycide (PLGA) biodegradable polymer. This study evaluated the in vivo degradation of the polymer coating, everolimus release time course, and vascular compatibility of the SYNERGY stent. METHODS AND RESULTS: SYNERGY stents were implanted in arteries of domestic swine. Devices were explanted at predetermined time points (up to 120 days) and the extent of PLGA coating or everolimus remaining on the stents was quantified. Everolimus levels in the arterial tissue were also evaluated. A pathological analysis on coronary arteries of single and overlapping stents was performed at time points between 5 and 270 days. PLGA bioabsorption began immediately after implantation, and drug release was essentially complete by 90 days; PLGA absorption was substantially complete by 120 days (>90% of polymer was absorbed) leaving a bare metal SYNERGY stent. Vascular response was similar among SYNERGY and control stents (bare metal, polymer-only, and 3× polymer-only). Mild increases in para-strut fibrin were seen for SYNERGY at an early time point with no significant differences in all other morphological and morphometric parameters through 270 days or endothelial function (eNOS immunostaining) at 90 or 180 days. Inflammation was predominantly minimal to mild for all device types. CONCLUSION: In a swine model, everolimus was released by 90 days and PLGA bioabsorption was complete shortly thereafter. The SYNERGY stent and its biodegradable polymer, even at a 3× safety margin, demonstrated vascular compatibility similar to bare metal stent controls.

Experimental evaluation of efficacy and healing response of everolimus-eluting stents in the familial hypercholesterolemic swine model: a comparative study of bioabsorbable versus durable polymer stent platforms.

BACKGROUND: The utility of animal models for the prediction of drug-eluting stent (DES) efficacy in human clinical trials is still unclear. The familial hypercholesterolemic swine (FHS) model has been shown to induce a human-like neointimal response to bare metal stent (BMS) implantation. However, its utility to discriminate efficacy signals following DES implantation is unknown. In this study, we aimed to test the efficacy and healing response of several everolimus-eluting stent (EES) platforms in the coronary territory of the FHS. METHODS: A total of 19 EES platforms (SYNERGY=6, SYNERGY½-dose=7, and PROMUS Element=6) and an identical BMS control (Element=6) were implanted into the coronary arteries of nine FHS. All implants were performed under intravascular ultrasound guidance using a 1.2 : 1 overstretch ratio. At 30 days, the vascular response to the implant was evaluated by quantitative coronary angiography, optical coherence tomography, and histology. RESULTS: At 28 days, all EES platforms showed a significant decrease in angiographic late lumen loss (between 27 and 37%) compared with the BMS control group. This finding was confirmed both by optical coherence tomography (mean neointimal thickness=28-42% reduction) and by histology (mean neointimal thickness=44-55% reduction). All EES platforms showed similar degrees of neointimal inhibition. The presence of moderate to severe para-strut inflammation was observed in 83% of the stent sections in the BMS group compared with 28.6% in the SYNERGY½-dose group and 0% in the SYNERGY and PROMUS groups (P=0.0002). There was a 68-95% reduction in MMP9 expression in the media in all EES platforms compared with the BMS controls. The presence of mild to moderate para-strut fibrin deposits ranged from 66.7 to 83.4% in all EES platforms compared with 16.7% in the EBMS group. CONCLUSION: The FHS coronary injury model showed the efficacy of several EES platforms compared with an identical BMS control. Everolimus eluted from different polymeric platforms showed lower levels of inflammation and slightly higher fibrin deposits compared with BMS controls.

Impact of stent surface on thrombogenicity and vascular healing: a comparative analysis of metallic and polymeric surfaces.

BACKGROUND: Emerging drug-eluting stent technologies are evolving toward the elimination of polymeric component used as the method for modulating drug delivery. Although this technological approach seems to be biologically appealing, the impact of durable polymers and metallic stent surfaces on vascular healing remains unclear. In the present study, we aimed to compare the independent effect of a durable polymer and a metallic stent surface on thrombogenicity and endothelial cell coverage using different in vitro and in vivo experimental models. METHODS AND RESULTS: Platinum chromium (PtCr) and polyvinylidene fluoride-co-hexafluoropropene (PVDF-HFP)-coated surfaces were evaluated in this study. Thrombogenicity was assessed by exposing all surfaces to human blood under shear flow conditions. The inflammatory potential of the material was evaluated by measuring cytokine release from THP-1 cells exposed to all surfaces for 24 hours. Endothelial cell coverage was evaluated by detection of CD31 after the stents were exposed to human coronary artery endothelial cells for ≤ 14 days. Platelet adhesion (P<0.01) and activation (P=0.03) on PVDF-HFP were greater than on PtCr. In vivo, PVDF-HFP revealed more neointimal area (P<0.01) and residual parastrut fibrin (P=0.01) at 30 days compared with PtCr. PtCr displayed higher endothelialization rates and higher vascular endothelial-cadherin expression at 7 and 14 days (P=0.02) compared with PVDF-HFP. CONCLUSIONS: Thrombogenicity and vascular healing differ among metallic and polymeric stent surfaces. PVDF-HFP exhibits higher degrees of platelet activation-adhesion and thrombus accumulation in vivo compared with PtCr. PtCr displayed higher degrees of endothelial surface coverage compared with PVDF-HFP surfaces.

Ontogenetic regulation of leukocyte recruitment in mouse yolk sac vessels.

In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.

Vasomotor function and re-endothelialisation after implantation of biodegradable abluminal polymer coated paclitaxel-eluting stents in rabbit iliac arteries: a time-course study.

AIMS: To evaluate the time-course of vasomotor function and re-endothelialisation after implantation of a novel platinum-chromium (PtCr) abluminal biodegradable polymer-coated paclitaxel-eluting stent (PES, Labcoat Element) in rabbit iliac arteries. METHODS AND RESULTS: Either PES (n=18) or an identical platform of bare metal stents (BMS, Element, n=18) were implanted in rabbit iliac arteries (six animals per time-point). At 14, 30, and 90 days, acetylcholine- and nitroglycerine-induced vasomotor reactivity at 5-10 mm distal to the stent was measured. Subsequently, the animals were terminated. The stented artery was bisected longitudinally for either SEM or en face CD31 immunochemistry examination. All arteries were patent with normal angiographic flow. Decreased endothelial-dependent vasomotion was found at both 14 and 30 days for PES compared to BMS (p<0.01, respectively); however, these differences resolved by 90 days. Endothelial-independent vasorelaxation was similar at all three time-points. Both SEM and en face staining demonstrated equivalent endothelial coverage on the surface of the stented segments above and between struts at all time-points. CONCLUSIONS: This novel bioabsorbable polymer abluminal-coated PES demonstrated vasomotor function comparable to BMS within three months post-deployment in the rabbit iliac model. Despite indistinguishable endothelial cell coverage on the stent surface between groups, earlier differences in vasomotion were detected: this finding suggests that the timing of restoration vasomotor function lags morphologic endothelial recovery.

Discovery of 2-[1-(4-chlorophenyl)cyclopropyl]-3-hydroxy-8-(trifluoromethyl)quinoline-4-carboxylic acid (PSI-421), a P-selectin inhibitor with improved pharmacokinetic properties and oral efficacy in models of vascular injury.

Previously, we reported the discovery of PSI-697 (1a), a C-2 benzyl substituted quinoline salicylic acid-based P-selectin inhibitor. It is active in a variety of animal models of cardiovascular disease. Compound 1a has also been shown to be well tolerated and safe in healthy volunteers at doses of up to 1200 mg in a phase 1 single ascending dose study. However, its oral bioavailability was low. Our goal was to identify a back up compound with equal potency, increased solubility, and increased exposure. We expanded our structure-activity studies in this series by branching at the alpha position of the C-2 benzyl side chain and through modification of substituents on the carboxylic A-ring of the quinoline. This resulted in discovery of PSI-421 with marked improvement in aqueous solubility and pharmacokinetic properties. This compound has shown oral efficacy in animal models of arterial and venous injury and was selected as a preclinical development compound for potential treatment of such diseases as atherosclerosis and deep vein thrombosis.

Characterization of the novel P-selectin inhibitor PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid] in vitro and in rodent models of vascular inflammation and thrombosis.

P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 microM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.

2-(4-Chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[H]quinoline-4-carboxylic acid (PSI-697): identification of a clinical candidate from the quinoline salicylic acid series of P-selectin antagonists.

P-selectin-PSGL-1 interaction causes rolling of leukocytes on the endothelial cell surface, which subsequently leads to firm adherence and leukocyte transmigration through the vessel wall into the surrounding tissues. P-selectin is upregulated on the surface of both platelets and endothelium in a variety of atherosclerosis-associated conditions. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of atherosclerosis. High-throughput screening and subsequent analoging had led to the identification of compound 1 as the lead candidate. Herein, we report the continuation of this work and the discovery of a second-generation series, the tetrahydrobenzoquinoline salicylic acids. These compounds have improved pharmacokinetic properties, and a number of them have shown oral efficacy in mouse and rat models of atherogenesis and vascular injury. The lead 31 (PSI-697), is currently in clinical development for the treatment of atherothrombotic vascular events.

Relative contributions of alpha4 and alphaL integrins to IL-4-induced leukocyte rolling and adhesion.

OBJECTIVE: To delineate the relative contributions of alpha4 and alphaL to mediate interleukin-4 (IL-4) induced leukocyte rolling, and the subsets of leukocytes that use these pathways to adhere. METHODS: Intravital microscopy was used to examine leukocytes in venules of cremaster muscles of mice receiving intrascrotal injections of IL-4. alpha4 and alphaL monoclonal antibodies (mAbs) were administrated either prior to (prophylactic) or 24 h following (therapeutic) treatment with IL-4. In addition, fluorescent microspheres coated with mAbs directed against CD4, CD8, or Gr-1 were injected into mice and the number of subset-specific adherent leukocytes was measured. RESULTS: Prophylactic inhibition of alpha4 and alphaL integrins prevented IL-4-induced leukocyte rolling flux (p< .05) and increased leukocyte rolling velocity twofold (p < .05), respectively, while blocking either integrin eliminated IL-4-induced leukocyte adhesion (p < .05). In contrast, therapeutic administration of both anti-alpha4 and anti-alphaL mAbs was necessary to completely inhibit IL-4-induced leukocyte adhesion (p < .05). Furthermore, CD8+ and Gr-1+ leukocytes utilized alpha4 and alphaL to adhere to postcapillary venules, whereas CD4+ leukocytes primarily utilized alpha4. CONCLUSIONS: Following tissue activation with IL-4, alpha4 and alphaL initiate the attachment and deceleration, respectively, of leukocytes during rolling, and are responsible for mediating the adhesion CD4+, CD8+, Gr-1+ leukocytes.